Outbreak of Cyclosporiasis in Korean Travelers Returning from Nepal

Cyclospora cayetanensis is an apicomplexan protozoan and is one of the most common pathogens causing chronic diarrhea worldwide. Eight stool samples with diarrheal symptom out of 18 Korean residents who traveled to Nepal were obtained, and examined for 25 enteropathogens including 16 bacterial species, 5 viral species, and 4 protozoans in stool samples as causative agents of water-borne and food-borne disease. Only C. cayetanensis was detected by nested PCR, and 3 PCR-positive samples were sequenced to confirm species identification. However, the oocysts of C. cayetanensis in fecal samples could not be detected by direct microscopy of the stained sample. As far as we know, this is the first report of a group infection with C. cayetanensis from a traveler visiting Nepal, and the second report of a traveler’s diarrhea by C. cayetanensis imported in Korea.

Molecular Prevalence and Genotypes of Cryptosporidium parvum and Giardia duodenalis in Patients with Acute Diarrhea in Korea, 2013–2016

Cryptosporidium parvum and Giardia duodenalis are the main diarrhea-causing parasitic pathogens; however, their prevalence in Korea is unknown. Here, we conducted a survey to determine the prevalence and genotype distribution of these 2 pathogens causing acute diarrhea in 8,571 patients hospitalized in 17 Regional Institute of Health Environment sites in Korea, during 2013–2016. C. parvum and G. duodenalis were detected and genotyped by nested PCR, and the isolate were molecularly characterized by sequencing the glycoprotein 60 (Gp60) and β-giardin genes, respectively. The overall prevalence of C. parvum and G. duodenalis was 0.37% (n=32) and 0.55% (n=47), respectively, and both pathogens were more prevalent in children under 9 years old. Molecular epidemiological analysis showed that the C. parvum isolates belonged to the IIa family and were subtyped as IIaA13G2R1, IIaA14G2R1, IIaA15G2R1, and IIaA18G3R1. Analysis of the β-giardin gene fragment from G. duodenalis showed that all positive strains belong to assemblage A. This is the first report on the molecular epidemiology and subtyping of C. parvum and G. duodenalis in such a large number of diarrheal patients in Korea. These results highlight the need for continuous monitoring of these zoonotic pathogens and provide a basis for implementing control and prevention strategies. Further, the results might be useful for epidemiological investigation of the source of outbreak.

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

Follow-up Study of Patients Previously Diagnosed with Lymphatic Filariasis in Korea

OBJECTIVES: Korea was an endemic area for lymphatic filariasis (LF), caused by the nematode parasite Brugia malayi, until the 1970s. The World Health Organization recognized Korea as LF-free in June 2008. However, it is necessary to confirm that patients that have had LF in the past still test negative, to prevent the re-emergence of LF in Korea. METHODS: We followed up a total of 83 patients who had been diagnosed with LF between 2002 and 2010 in endemic LF areas. RESULTS: Fifty-two of the 83 subjects were negative for LF, whereas 31 subjects had re-located to a different city or province, were dead, or were unaccounted for. Most subjects with negative test results still exhibited edema in the legs or the arms, and some complained of redness and swelling in the legs or ankle joints. However, we found that these symptoms were due to diseases other than LF. CONCLUSION: In this follow-up study, we did not find any evidence indicating the potential re-emergence of LF in Korea.

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.